J Assist Reprod Genet (2014) 31:1115–1137 DOI 10.1007/s10815-014-0280-6

REVIEW

A multi-faceted approach to understanding male : mutations, molecular defects and assisted reproductive techniques (ART)

Eisa Tahmasbpour & Dheepa Balasubramanian & Ashok Agarwal

Received: 11 March 2014 /Accepted: 16 June 2014 /Published online: 13 August 2014 # Springer Science+Business Media New York 2014

Abstract Introduction Background Theassistedreproductivetechniquesaimedto assist infertile couples have their own offspring carry signifi- Human male infertility is a major health problem that affects cant risks of passing on molecular defects to next generations. approximately 10 to 15 % of couples worldwide [1, 2]. Spe- Results Novel breakthroughs in gene and interactions cifically, in the Western countries, 15 % of couples are diag- have been achieved in the field of male infertility using nosed with male infertility [3]. In about 50 % of these cases, genome-wide proteomics and transcriptomics technologies. the underlying problem lies with the male, either solely or in Conclusion Male Infertility is a complex and multifactorial combination with female factors [4]. Male infertility is a disorder. multifactorial syndrome encompassing a wide variety of dis- Significance This review provides a comprehensive, up-to- orders [5]. Anatomic defects underlying male infertility in- date evaluation of the multifactorial factors involved in male clude varicocele, vesicular damage due to torsion, obstruction infertility. These factors need to be first assessed and under- of testicular sperm passage and ejaculatory failures, genital stood before we can successfully treat male infertility. tract infections, gametogenesis dysfunction, molecular genet- ics disorders, endocrine disturbances and immunologic prob- lems [4, 6]. Additionally, factors such as life style, environ- Keywords Male infertility . Spermatogenesis . ment [7, 8] and smoking [9]havealsobeenreportedtoaffect Ychromosome. AZF region . Molecular genetics . gamete and embryo development. Infertile men with no past Epigenetics history and normal semen analyses are designated as ‘idio- pathic infertile’. Several factors including oxidative stress induced by reactive oxygen species (ROS), sperm DNA dam- Eisa Tahmasbpour and Dheepa Balasubramanian have contributed age and molecular genetic abnormalities are responsible for equally the symptoms of Idiopathic infertility [10–13]. The most Capsule This review reports the genetic, molecular and phenotypic common causes of male infertility are summarized in Table 1. factors that greatly facilitate the understanding and treatment of a multi- Genetic abnormalities leading to male infertility are respon- factorial disorder, Male infertility. sible for 15 to 30 % of cases, with a prevalence of Y chromo- E. Tahmasbpour some abnormalities [14, 15]. Whereas many other genetic Department of Biology, Islamic Azad University, Sari, Iran abnormalities that cause male infertility are still unknown, it D. Balasubramanian is likely that most cases of idiopathic infertility could be Case Western Reserve University, 44106 Cleveland, OH, USA accounted for by underlying genetic causes [16]. Molecular defects and genetic alterations responsible for male infertility * A. Agarwal ( ) disrupt physiological processes including hormonal homeo- Center for Reproductive Medicine, Cleveland Clinic, 44195 Cleveland, OH, USA stasis, spermatogenesis and sperm quality [17, 18]. Chromo- e-mail: [email protected] somal abnormalities, single gene point mutations including 1116 J Assist Reprod Genet (2014) 31:1115–1137

Table 1 Distribution of the most common causes of male infertility somal disorders) are the cause of their infertility, then there is Male infertility disorders Percentage References an increased risk of transmitting the genetic defects to future generations. Also of importance to note is that it is becoming Varicocele 37–40 % [221] increasingly clear that abnormalities, both qualitative and Endocrine disorders >20 % [222] quantitative, in semen analysis might be the ‘presenting symp- Genital tract infections 8–35 % [223] tom or phenotype’ of a variety of pathologies that can also Testicular failure 9 % [224] affect non-reproductive organs (Fig. 1). In following sections, Genetic disorders 15–30 % [1] we will discuss important molecular and genetic causes of Antisperm antibody 8–19 % [225] human male infertility and describe the state-of-the art tech- Idiopathic 15–25 % [226] nologies utilized to investigate this disorder, as well as high- light the increased need for genetic counseling and testing. mutation of cystic fibrosis transmembrane receptor gene (CFTR), polygenic or multifactorial genetic defects including Genetic causes of male infertility Y deletions or micro deletions, mitochondrial DNA (mtDNA) mutations, endocrine disorders of genetic Chromosomal abnormalities origin are the most frequently reported genetic causes of male infertility (Table 2). In addition, mutations and polymor- Chromosomal defects are very common in infertile men, with phisms of various involved in spermatogenesis and a reported incidence of 2–15 %. The chromosomal aberrations other aspects of male reproduction have also been found to involve the sex in 3.8 % cases and the auto- be associated with male infertility [19]. somes in 1.3 % of cases [20]. In addition to sex or autosomal With recent advances in assisted-reproductive technologies chromosome abnormalities, numerical and structural defects such as, in vitro fertilization (IVF) and intracytoplasmic sperm of chromosomes also abound in infertile males. Numerical injection (ICSI), it is possible to offer reproductive hope to disorders result from changes in the normal number of chro- men once considered to be irreversibly sterile. However, in mosomes per cell. Chromosomal aneuploidy is a genetic these instances, if genetic anomalies (Mendelian and chromo- condition in which an individual has either an abnormal

Table 2 Prevalence and phenotype of the most common genetic anomalies associated with male infertility

Genetic abnormalities Phenotype Prevalence (%) References

Chromosome aberrations Azoospermia to normospermia 2–10 % [1] Numerical disorders Klinefelter’s syndrome Azoospermia to severe oligospermia 5–10 % azoospermia 2–5 % oligospermia [1] Other sex chromosomes Azoospermia to normospermia 0.1–0.2 % [20] Structural disorders Robertsonian translocations Azoospermia to severe oligospermia 0.5–1% [20] Reciprocal translocations Azoospermia to severe oligospermia 0.5–1% [32] Y chromosome deletions and microdeletions Azoospermia to severe oligospermia 5–10 % [226] AZFa Azoospermia to SCOS 0.5–1% [34] AZFb Azoospermic to arrest of spermatogenesis 0.5–1% [34] AZFc Azoospermia to severe oligospermia 3–7% [20] AZFb,c SCOS to arrest of spermatogenesis 0.5–1% [34] Partial deletions of AZFc Azoospermia to normozoospermia 3–5% [34] Genetic mutations CFTR Obstructive azoospermia 4–5% [20] AR Azoospermia to oligospermia 2–3% [82] KAL-1 Hypogonadism hypogonadotropic 5 % [18] INSL3-LGR8 Cryptorchidism 4–5% [227] J Assist Reprod Genet (2014) 31:1115–1137 1117

Fig. 1 Common genetic, molecular and epigenetic factors contributing to the development, and the diagnostic and preventive measures to treat male infertility

number of one or more chromosomes, or has fragments of Although most patients with KS are infertile, there have chromosomes lacking, or in excess. The severity of the been few patients, typically in mosaic cases (46, XY/47, phenotypic manifestations depends on which chromosomes XXY), who have conceived normally without assisted medi- are affected [21]. Structural disorders include deletions, dupli- cal technology [24]. Within the testis of azoospermic men cationsortranslocationsofchromosomes,aswellaschromo- with KS, sperm can be retrieved in approximately 50 % of somal rearrangements. cases on testicular exploration from focal areas of spermato- genesis. In such cases, ICSI can be performed with an esti- Klinefelter’s syndrome (KS) mated success rate ranging from 30 to 50 % [4, 24]. In light of these findings, testicular sperm extraction and ICSI may be Klinefelter’s syndrome (47, XXY) is the major sex considered a potential option to attain pregnancy in males with chromosomal/numerical defect detected in newborn males azoospermia and KS patients [24]. Preimplantation genetic (0.1–0.2 %) [22]. The prevalence of KS among infertile men diagnosis using FISH techniques is another option that has is very high, up to 5 % in severe oligozoospermia and 10 % shown promise in selecting normal embryos from KS men. In in azoospermia [23]. This karyotype arises spontaneously the future, it can be visualized that early sperm retrieval and when paired X chromosomes fail to disjoin in the first or sperm banking would be highly recommended for men with second phase of meiosis during oogenesis or spermatogen- KS [25]. esis. Extra copies of genes on the X chromosome in these patients interfere with male sexual development and pre- Chromosomal translocations vents the testes from functioning normally, consequently reducing the levels of testosterone that are responsible for Robertsonian and reciprocal translocations are the most fre- the impaired spermatogenesis [4]. quently observed structural chromosomal abnormalities in 1118 J Assist Reprod Genet (2014) 31:1115–1137 humans [26]. Although the prevalence rate of robertsonian Y chromosome and male infertility translocation (RT) is 0.9 in infertile males, this figure is 9 times higher than in the general population [27, 28]. Recipro- Extensive research has been performed on the association of Y cal translocations are observed in 0.9 of 1000 newborns and chromosome deletions with male infertility, since it contains involve exchange of two unrelated chromosomal segments several genes that are critical for spermatogenesis and the [29]. They have been found in approximately 1 % of infertile development of male gonads. The prevalence of Y chromo- men and commonly occur in azoospermic men compared to some deletions is estimated to be ~1:2000 to 1:3000 males. It oligozoospermic males [16, 30]. Robertsonian translocations represents the second most common genetic cause of could potentially affect male fertility and pregnancy by alter- spermatogenetic failure in infertile males after Klinefelter’s ing the pattern of the spermatozoa at different syndrome [18, 37]. The incidence of Y micro deletions in stages [31, 32]. This is very evident in oligozoospermic infertile men varies from 1 to 55 % [38, 39]. (1.6 %) and azoospermic (0.09 %) men [30]. In all carriers, The Y chromosome is divided into three regions, the reciprocal translocations have impacted spermatogenesis, proximal, middle, and distal Yq11 and is designated as AZFa, demonstrated by poor sperm quality or sperm aneuploidies, AZFb and AZFc (Fig. 2), after the azoospermia factor AZF severe azoospermia and infertility. Although carriers of RT [40, 41]. Further, a fourth AZF region that overlaps with may exhibit a normal phenotype, spermatogenesis in male AZFb and AZFc regions is termed as AZFd [42]. These carriers of RT is variably affected, ranging from normal to regions contain important genes, notable for their role in germ oligoasthenospermia or azoospermia [17, 30]. It is thereby of cell development. Furthermore, these regions are of greater paramount importance to analyze the chromosomal constitu- significance since studies have revealed that gene deletions in tion in the sperm of RT carriers, in order to provide preim- these regions have been shown to be pathogenically involved plantation genetic diagnosis and adaptive genetic counseling in male infertility, specifically, azoospermia or severe oligo- to persons undergoing IVF treatment. zoospermia [43]. Increased awareness has created increased demand for Yq deletion screening. Y micro deletion screening is strongly Other sex chromosomal aneuploidies recommended for infertile men with severe sperm defect of unknown origin (ranging from azoospermia to oligozoosper- In addition to these numerical chromosome abnormalities, mia). Although, ART techniques such as ICSI, testicular men with karyotypes 47XYY, mosaic 45X/46XY, 45× and sperm extraction (TESE) and IVF now facilitate infertile 46XX also occur but their fertility status is variable [1]. A 47, men associated with Y-chromosome micro deletion to achieve XYY male birth occurs in approximately 1:1000 live births. pregnancies [44], there is concern that such pregnancies may Although they are phenotypically normal, this karyotype can produce male offspring with similar micro deletions and con- also result in infertility [16]. In patients with spermatogenic sequent infertility [45]. In the following sections, we will failure, maturation arrest or the Sertoli-cell-only syndrome discuss the different AZF regions of the Y chromosome and (SCOS) with associated hormonal abnormalities and eventu- the functional significance of the genes they harbor, in detail. ally oligospermia or azoospermia may be observed [21]. The mosaic karyotype 45×/46XY is found in infertile men with an The AZFa region incidence of 4 % and known to cause infertility [33]. All males with discordant sex chromosomal pattern are azoospermic due The AZFa locus is located at the proximal portion of long arm to absence of long arm of the Y chromosome containing the of the Y chromosome (Fig. 2) and spans about 1.1 Mb [46]. azoospermic factor (AZF) gene, which is necessary for normal This region includes 4 genes including USP9Y (Ubiquitin- spermatogenesis [34]. Translocation of SRY gene (testis- specific protease 9, Y chromosome), DBY (dead box on the determining factor) from chromosome Y to chromosome X Y), UTY (ubiquitous TPR motif on the Y) and TB4Y (thymo- hasalsobeenreported[35]. In a study by Abusheikha et al., sin B4-isoform). Deletion of the candidate genes, USP9Y and [36], they reported a male with normal male phenotype, in DBY are considered to be responsible for infertility. USP9Y is whom seminal analysis showed complete azoospermia. More- present as a single copy gene that is expressed in a wide over, polymerase chain reaction analysis of genomic DNA variety of tissues and functions as an ubiquitin hydrolase failed to detect the presence of the SRY gene. Men with [47]. DBY encodes an RNA helicase, is ubiquitously azoospermia and karyotype 45, X have also mutations in expressed and is the major gene located in AZFa region. AZF gene. A comprehensive investigation detailing the fre- While deletions or small mutations in USP9Y seem to be quency, etiology and diagnosis of these rare syndromes is associated with severe hypo-spermatogenesis only, patients thereby necessary for these patients to receive gentle manage- with deletions of DBY may present with a combination of ment and counseling through a cooperative interdisciplinary the two: sertoli cell-only syndrome (SCOS), a condition char- approach [36]. acterized by the presence of complete Sertoli cells in the testes J Assist Reprod Genet (2014) 31:1115–1137 1119

Fig. 2 A schematic picture of Y chromosome displaying AZF regions (AZFa-c) and associated genes. Enlarged portion of AZFc region illustrates the discussed microdeletions. (a) Normal AZFc region, (b) gr/gr deletion, (c) b1/b3 deletion, and (d)b2/b3 deletion

but a lack of spermatozoa in the ejaculate and severe hypo- is arrested completely, indicating that RBMY and PRY are the spermatogenesis [17, 48]. On the contrary, patients with dele- major genes involved in fertility [17, 34]. tions of both USP9Y and DBY seem to invariably demonstrate Other genes including SMCY, CDY2, XKRY, HSFY, azoospermia with a testicular histology of SCOS [17, 49]. LOC170324, CYorf14, CYorf15A/B, TTY5, TTY6, TTY9, Deletion of the USP9Y gene can additionally cause azoosper- TTY10, TTY13, TTY14, XKRYand RPS4Y2 have been mapped mia, oligozoospermia, or oligoasthenozoospermia [15, 50]. to this region, but their role in the spermatogenic process is not well elucidated yet [1, 54]. Recent evidence points to a role of CDY2 in male germ cell development, specifically in the The AZFb (P5/proximal-P1) region to protamine transition [20, 55]. HSFY encodes for a heat shock factor type DNA binding domain and is predicted The AZFb locus spans about 3.2 Mb of the Y chromosome to bind to heat shock promoter elements, as a transcriptional long arm (Fig. 2) and includes two important genes, EIF1AY activator [54]. Mahanta et al.[55] reported that the frequency (translation initiation factor 1 A, Y isoform) and RBMY (RNA of Y chromosome micro deletions in XKRY gene is 23.1 %. binding motif on Y) critical for male infertility. EIF1AY en- Although very little information exists for other AZFb genes, codes a ubiquitously expressed translation initiation factor. there is sufficient evidence to suggest that that five single copy The other main gene in the AZFb region is RBMY.Approxi- genes (LOC170324, SMCY, EIF1AY, RPS4Y2, and GAPD- mately 15–30 copies of these genes are dispersed in the short similar), and two duplicate genes (HSFY and LOC14007/ and long arm of the Y chromosome [51]. RBMY1 encodes a 140020) are deleted in some infertile patients [56]. testis-specific RNA binding protein/splicing factor that is expressed in the nuclei of spermatogonia, spermatocytes, The AZFc (b2/b4) region and round spermatids [52]. Deletions of the AZFb region include at least one functional copy of the RBMY1 gene. A The AZFc region spans about 3.5 Mb of the long arm of Y family of PRY genes, essential in the regulation of apoptosis is chromosome (distal Yq11) and contains testis-specific genes also found in the AZFb region [53]. Whereas small AZFb involved in male spermatogenesis (Fig. 2)[57]. Deletions of deletions have been related to variable testicular phenotypes the AZFc locus occur more frequently (65–70 %) than AZFa [54], larger AZFb deletions and those that include genes or AZFb locus [15]. Deletions of the AZFc locus manifest in PRY1/PRY2 seem to cause complete meiotic arrest [34]. Re- 10–20 % of infertile men [58] and specifically in cases of cently, studies have revealed that in cases where all the genes moderate oligozoospermia (0.7 %), severe oligozoospermia in the AZFb region excluding RBMY and PRY are deleted, (4–14 %), or secretory azoospermia (11–18 %) [41]. In addi- patients present with hypo spermatogenesis [20]. However, if tion, AZFc deletions are associated with testicular histologies both the RBMY and PRY genes are removed, spermatogenesis varying from hypo-spermatogenesis (HP) to MA or SCOS 1120 J Assist Reprod Genet (2014) 31:1115–1137

[59]. Recently, studies have demonstrated that although only very high incidence of AZFc deletions has been demonstrated the AZFa and AZFb regions are required to initiate spermato- in incomplete SCOS. genesis, without the AZFc region, spermatogenesis will not be Another important gene harbored in the AZFc region is completely normal [29]. CDY1, which is involved in the replacement of The genomic structure of AZFc region is composed mainly during spermatogenesis [52]. Previously, it was revealed that of large repeats of sequence blocks termed “amplicons”,or- loss of CDY1 variants (a/b) is significantly higher in infertile ganized into palindromic structures that show high (>99.9 %) men than control men. But, in another conflicting study, no sequence identity between the arms [60]. Such structures may association between DAZ1/2, DAZ3/4, CDY1a,orCDY1b and undergo frequent inversion and gene-conversion events [61], spermatogenic failure was detected [74]. Screening for AZF as well as duplications and deletions [62]. These small partial micro deletions before undergoing ART is mandatory and deletions or sub-deletions produce a wide array of phenotypes, very critical for azoospermic and severely oligozoospermic ranging from normospermic to azoospermic, aided by their patients, since it is well established that their male offspring interaction with the environment and their genetic background will inherit the Yq micro deletions and infertility as well [75]. [17]. The gr/gr, b1/b3, and g1/g3 (b2/b3) are the most frequent Associations between other Y chromosome genes including sub deletions in the AZFc region of the Y chromosome [63] SRY (Sex Determining Region) and TSPY (testis-specific pro- (Fig. 2). It is distressing to note that these rearrangements or tein Y-encoded) and male infertility have also been recently polymorphisms can be transmitted to their male progeny and established. Deletion of the SRY gene occurs in 15 % of 46 may represent a risk factor for decreased testis function and XY sex reversed females [76]. Additionally, mutations in SRY male sub fertility. However, in a study by Hopps et al., they gene have also been reported in 10–15 % of 46 XY males with reported that the majority of men with AZFc deletion have gonadal dysgenesis [77]. Recent studies support the link be- sperm within the semen or testes available for use in IVF/ICSI tween SRY alterations, gonadal dysgenesis and primary infer- [64]. tility [78]. Evidence to signify TSPY’s involvement in The b1/b3 deletion removes the PRY genes and reduces the gonadoblastoma and prostate has been reported [79]. copy numbers of several other genes. However, the prevalence of the b1/b3 deletion in the human population is very low and Autosomal and X-linked genes mutations its frequency varies, with only 18 deletions published to date [65]. Its effect on spermatogenesis is unknown [66]. The b2/ In addition to sex chromosomal genes, many autosomal and b3 deletion removes 1.8 Mb of AZFc or DAZ3/DAZ4 genes X-linked genes are also necessary for normal sexual develop- and was detected only in men with spermatogenic failure [65]. ment, testis determination, testis descent, spermatogenesis and The gr/gr deletion [67, 68] removes half of the AZFc region eventually fertility in men. Given the importance of these including two copies of the DAZ gene family (DAZ1/DAZ2), genetic regions, it is not surprising that mutations, transloca- one copy of the CDY1 gene family (CDY1a) and one copy of tions, deletions or inversions that alter these regions may result the BPY2 gene [66]. The gr/gr deletion event occurs with a in severe infertility and azoospermia. The human X chromo- relatively high frequency (3.5 %) in different Y haplotypes some is enriched with testis-specific genes that may be crucial among men with spermatogenic failure [66]. It varies from 2.1 for spermatogenesis and male fertility. Mutations in autosomal to 12.5 % among all cases, and from 0 to 10.2 % among CFTR, c-kit receptor (c-kitR), KAL-1, INSL3-RXFP2 and X- normozoospermic controls [69]. In a conflicting report by linked androgen receptor (AR) genes are most common and Eloualid et al., [65] they have reported that gr/gr deletions lead to infertility due to defects in germ cell proliferation or occur at similar frequencies of 5.83 and 6.25 % in patient and urogenital system. We will summarize the role of some auto- control populations respectively, suggesting that these dele- somal and X linked genes in spermatogenesis and the effects tions are not associated with spermatogenic failure. of different mutations or polymorphisms on male infertility in Within the AZFc region, substantial evidence points to the the following sections. importance of DAZ gene family in normal spermatogenesis. Deletion of DAZ accounts for 10 % of cases of men with TheCFTRgenemutations spermatogenic defect [70, 71]. Several studies have identified a single nucleotide polymorphism of DAZL that confers sus- Cystic fibrosis (CF) is a heterogeneous genetic disease caused ceptibility to defects in spermatogenesis [72]. The deletion of by mutations in CFTR gene, located on chromosome 7 the DAZ1/DAZ2 doublet, as observed in a DAZ gene copy- (7Q31.2). There is a positive correlation between CF and specific deletion analysis, was considered responsible for congenital bilateral absence of the vas deferens (CBAVD), a severe oligozoospermia [73]. The importance of the DAZ1/ form of male infertility that is a frequent cause of obstructive DAZ2 doublets was also highlighted when deletion of azoospermia. This suggests a direct association of CFTR DAZ1/DAZ2 was seen in incomplete MA and was responsi- mutations and poor sperm quality in healthy men with CF ble for the testicular phenotype of residual spermiogenesis. A and CBAVD. More than 95 % of adult men with CF are J Assist Reprod Genet (2014) 31:1115–1137 1121 infertile because of obstructive azoospermia. Additional mu- oligospermia [83]. It is important to recommend and offer tations include the 5 T polymorphism within intron 8 in the screening for AR mutations and appropriate pre-conception CFTR gene that was found in 21 % of the subjects and is counseling before testicular sperm aspiration for ICSI in considered a mild mutation, specifically associated with male azoospermic men, at risk of having AR mutations [85]. sterility [43, 80]. The 7 T polymorphism within intron 8 and the missense R117H mutation within exon 4 in the CFTR gene The INSL3-LGR8 genes mutations were also reported [4, 81]. Other common mutations including G542X, G551D, R553X, W1282X and N1303K occur with a Insulin-like 3 growth gene (INSL3), also known as relaxin-like frequency of 1–2 %. Recently, studies have reported that factor (RLF) or Leydig insulin-like protein (LEY I-L), local- CFTR gene mutations are a relatively frequent cause of male ized on chromosome 19 and its receptor LGR8 (leucine-rich- infertility. Although techniques including IVF and ISCI have repeat-containing G protein-coupled receptor 8), localized on facilitated CBAVD patients to father children, the couples chromosome 13 are both functionally involved in the molec- have an increased risk of bearing a child with CF. Thereby, ular mechanism of testis descent from abdomen to scrotum it is imperative to advocate genetic testing and counseling for during fetal development [1]. Cryptorchidism is one of the these couples [43]. Nowadays, PGD has been regarded as a most frequent (2 % of male births) congenital abnormalities in useful tool to identify the presence of CFTR mutations in humans and causes non-obstructive azoospermia in 20 % of patients. cases [4]. It can result in male infertility due to impaired spermatogenesis. Reduced sperm concentration, increased The androgen receptor (AR) gene mutations FSH and reduced inhibin B plasma levels that correlate with reduced testicular volume in adult life have also been reported, Androgens (testosterone (T) and 5α-dihydrotestosterone even after treatment by orchidopexy [4]. Recent data have (DHT)) are critical steroid hormones that determine the de- reported that mutations in INSL3 gene or LGR8/GREAT occur velopment and maintenance of spermatogenesis. Perturba- in approximately 5 % of men with cryptorchidism [86]. How- tions in their function can result in spermatogenic failure and ever, there is conflicting evidence to believe that these fre- male infertility. Mutations in AR gene, an X-linked genetic quent pathologies could possibly be caused by mutations in condition causes male infertility with a frequency of 1:60,000 other genes as well [1]. of live deliveries. In infertile men, the AR mutations are believed to affect at least 2 % of patients [82]. Recent studies The Kallmann 1 (KAL-1) gene mutations have shown that mutations or polymorphisms in AR gene and its expressed protein may also result in depressed spermato- Kallmann syndrome (Ks) is an X-linked recessive disease that genesis and idiopathic male infertility, without any abnormal- is characterized by isolated hypogonadotropic hypogonadism ities in male secondary sexual characteristics [83]. (HH) and infertility. It is a rare genetic condition that accrues Mutations in TAD and LBD subunits of AR are probably in approximately 1 in 10,000–60,000 live births (1:10000 in most common. About 19 mutations in the TAD are reported to men and 1:50000 in women). Most patients have be associated with some form of androgen insensitivity, in gonadotropin-releasing hormone (GnRH) deficiency. Clini- which patients have a decreased sperm count and a high cally, it is characterized by low serum concentrations of tes- percentage of abnormal sperm [82]. A critical region known tosterone (T), low levels of luteinizing hormone (LH) and as CAG-nucleotide repeats exists in TAD exon and contains follicle-stimulating hormone (FSH) with normal prolactin an average of 21±2 repeats. It is polymorphic and reported to levels, as well as delayed puberty, small testes, incomplete be either increased or decreased in infertile men. CAG- sexual maturation, lack of secondary sexual features, erectile nucleotide repeats are also reported in TAD exon but exami- dysfunction and absence of ejaculation, anemia and osteopo- nation of its polymorphism has produced limited evidence. rosis and sometimes even cryptorchidism [87, 88]. Further- Data from some studies have established that AR missense more, testicular pathology displays a wide array of findings substitutions contribute about 2 % and CAG repeat expansion from SCSO to focal areas of complete spermatogenesis. Male (>28 repeats) may contribute up to 35 % of male infertility infertility in these cases is treatable with gonadotropins (LH cases [82]. A preliminary research indicated that the mean AR and FSH) replacement over 12 to 18 months, which induces gene CAG repeat length was significantly larger in sperm in the ejaculate in 80 % of men. azoospermic subjects than in control fertile men [84]. Muta- Although in about 75–80 % of Ks patients, the causative tions in TAD range from point mutations, insertions or dele- gene is unknown, mutations in several genes including KAL1, tions, or altered CAG repeats of AR gene. Mutations in AR FGFR1 (Fibroblast growth factor receptor 1), PROKR2 LBD have also been reported. Moreover, genetic screening of (Prokineticin receptor 2), PROK2 (Prokineticin 2), CHD7 infertile males has also revealed several loci in the AR LBD (Chromodomain helicase DNA binding protein) and FGF8 that are associated with reduced testicular volume and severe (Fibroblast growth factor 8) that are predominantly inherited 1122 J Assist Reprod Genet (2014) 31:1115–1137 in an autosomal dominant manner, are known to be associated SHBG levels were associated with higher androgen availabil- with Ks [89]. Although several genes mutations are responsi- ity and thus increased levels of spermatogenesis. In particular, ble in Ks, the discovery of the causes is clinically relevant they found that short AR (with <22 CAG repeats) were because it can enable clinicians to better understand the de- associated with lower sperm motility, while long AR alleles velopment of the neuroendocrine axis. were associated with higher sperm motility. However, no effect of the AR (CAG)n polymorphism on sperm concentra- The MTHFR (methylenetetrahydrofolate reductase) gene tion was observed in accordance with previous studies. These mutations results support that these genes might contribute to sperm’s basic parameters. Furthermore, this could provide an expla- The enzyme 5-methylenetetrahydrofolate reductase nation of how SHBG collaborates with AR to influence sperm (MTHFR), encoded by MTHFR gene, is a key enzyme in quality, by affecting intra-testicular androgen levels. folate metabolism. Many studies have shown that folate defi- ciency is common in infertile men and the related hyperho- The estrogen receptors (ESR) genes mutations mocysteinemia is a risk factor for spermatogenesis deficiency and male infertility [89]. The MTHFR enzyme deficiency and The role of estrogens (ES) in regulation of male reproductive hypo-methylation may inhibit gene expression, cause absence functions and testicular development is widely accepted [95]; of germinal cells and spermatogenesis arrest [90]. Recent however the molecular mechanism of their physiological role studies have reported that mutations in MTHFR gene have a in spermatogenesis is not clearly defined. The ES signaling is negative effect on male infertility. Among the significant known to be mediated by at least two functional isoforms of mutations studied, C677T (alanine for valine) is most com- estrogen receptors (ER) known as ERα and ERβ that are mon in infertile men with MTHFR deficiency [91, 92]and encoded by two different genes on the long arm of chromo- causes a decrease in the activity of MTHFR enzyme causing somes six (6q25) and 14 (14q23-24), respectively [22]. In dysregulation of folic acid metabolism, hyperhomocysteine- human testis, ERα and ERβ were also found in ejaculated mia, errors in the methylation of genomic DNA and subse- spermatozoa, early meiotic spermatocytes and elongated quent implications in the expression of spermatogenesis genes spermatids-independent ERα signaling is important for con- induced by under methylation. Singh et al., [93]reportedthat centrating epididymal sperm, whereas estrogen-mediated the homozygous (C/C) A1298C polymorphism of the ERβ signaling is essential for germ cell progression or viabil- MTHFR gene was also present at a statistically high signifi- ity [96]. The ER genes thereby represent a logical target for cance in idiopathic azoospermic infertile men. The findings mutational analysis in the infertile male. Although some work suggest that C677T and 1298CC homozygosity are an addi- evidenced an association between estrogen insufficiency and tional risk for molecular genetics of male infertility. Infertile abnormal spermatogenesis, little attention has been paid to the patients with hyperhomocysteinemia should be advised to role of ER gene mutations or polymorphisms in male screen for C677T mutation and simultaneously recommended infertility. a folate-rich diet or folate supplements. In the recent past, a possible link between ERα polymor- phisms and male infertility has been reported in Greek and The sex hormone–binding globulin (SHBG) gene mutation Japanese populations [97, 98]. In knockout models, removing the ERα (αERKO) or the aromatase genes (ArKO) showed The human sex hormone–binding globulin (SHBG) that is deficiency in the process of spermatogenesis and subsequent- expressed in testicular germ cells contributes to the concen- ly, reduced epididymal sperm count, sperm motility and fer- tration of androgens in the testis by binding or increasing tilizing ability [99]. Recently, the genetic screening of the androgen bioavailability [46]. Recently, the SHBG gene locat- ERα gene locus has revealed the existence of several poly- ed on chromosome 17 (17p13.1), has also been studied for a morphic sites including the PvuII (T397C) and XbaI restric- possible role in spermatogenesis and male infertility. The tion fragment length polymorphisms (RFLPs) in intron I and SHBG expression potentially influences SHBG levels in hu- the (TA)n variable number of tandem repeats (VNTR) within man sperm and consequently androgen bioavailability. In the promoter region of the gene [21]. In a study by Guarducci addition, the fact that SHBG accumulates between the outer et al., [100] they investigated the (TA)n repeat polymorphism acrosomal membrane and the sperm plasma membrane em- situated in the promoter region of the ERα gene in a large phasizes the functional significance of SHBG expression in group of infertile and normospermic men. Although the (TA)n male reproduction [47]. polymorphism failed to show a significant association with In a study by Lazaros et al., [94], they investigated the male infertility, they found a significant effect of this poly- possible role of SHBG (TAAAA) n polymorphism on male morphism on sperm count. They suggested that specific allelic factor fertility. An association was found between SHBG combinations of the ERα that confer a stronger estrogen effect (TAAAA) n polymorphism and sperm concentration. Higher may negatively influence human spermatogenesis. J Assist Reprod Genet (2014) 31:1115–1137 1123

On the other hand, the ERβ knockout mice were fertile, but testes in the preliminary stages of spermatogenesis. USP26 showed prostate and bladder hyperplasia [101]. Aschim et al., gene has emerged as an attractive novel candidate in the study [102] reported that the RsaI polymorphism in the ERβ gene of male infertility. Recent data have indicated increased num- appears to be associated with infertility, showing a three times ber of mutations and SNPs in the USP26 gene in men with higher frequency of the heterozygous RsaI AG-genotype in severe male factor infertility. Zhang et al., [107]investigated the infertile group compared with controls. In contrast, in the incidence of SNPs in USP26 gene and its involvement in another study by Khattri et al., [103], they found no significant idiopathic male infertility in China. They showed three vari- correlation in the incidence of the known SNPs or novel ations including a compound mutation (364insACA; 460G> mutations between infertile and fertile men. Moreover, infer- A) and a 1044 T>A substitution in azoospermia, oligozoos- tile men having ERβ mutations had normal reproductive tract permia and asthenozoospermia patients. All three variations and serum hormone levels. They postulated that the SNPs and led to changes in the coding amino acids. In a subsequent mutations in ERβ gene are not a common cause of spermato- study, Zhang et al., [108] concluded that 22 % of infertile genesis failure in Indian men, although mutations specifically patients exhibited changes in the USP26 gene (364insACA found in infertile men can affect , translation or and 460G>A (19.5 %) and 1044 T>A substitution (2.4 %)). have synergic effect with other variants in causing infertility Lee et al., [109] examined the association between some [104]. USP26 alleles and haplotypes with spermatogenic defect in Taiwanese and Chinese populations. In that study, they dem- The FSH/LH receptors (FSHR/LHR) genes mutations onstrated that the frequencies of five SNPs (370- 371insACA, 494 T>C, 576G>A, ss6202791C>T, 1737G> The follicle stimulating hormone (FSH) and luteinizing hor- A) were significantly higher in infertile patients than control mone (LH) regulate spermatogenesis. Although mutations in subjects. Although some USP26 alleles and haplotypes were the LH or FSH receptor gene are rarely seen, a number of found in the populations, the haplotype TACCGA was the SNPs in the LHR gene and LH gene are associated with most common (28 %) in patients with spermatogenic defect. variable pathologies of the male gonad [21]. The SNPs give Other novel variants such as 393A>T, 468 T>G, 520 T>G, rise to different FSHR and LHR haplotypes that modify the 565 T>G, 2144A>T, 2182A>T, 2195 T>C, 2204 T>G, action of FSH and LH. Mutations in the LHR gene lead to 2239A>T, 2247A>C, 2250A>G and 2271 T>C were also hypogonadal phenotypes (Leydig cell hypoplasia) because of linked to impaired spermatogenesis. Recently, Ribarski et al., a delay in fetal and pubertal male development. Mutational [110], reported high frequency (4.7 %) of 1090C>T mutations screening of the FSHR gene revealed several SNPs of the in infertile patients. Stouffs et al., [111] reported the presence FSHR gene, 1 in the promoter region at nucleotide position- of some mutations in USP26 gene such as 370-371insACA, 29 (G to A exchange) and 2 in exon 10 including positions 494 T>C and 1423C>T in patients (7.2 %) with SCOS. 680 and 307 [105]. Furthermore, variable suppression of All this data is underpins the importance of USP26 gene in spermatogenesis and fertility was diagnosed in men homozy- male reproduction and spermatogenesis. Although these stud- gous for the inactivating A189V mutation in exon 7 of the ies suggested that a specific genetic cluster might be associat- FSHR gene [1]. Conflicting studies reported a different allelic ed with testicular dysfunction, further studies based on a larger frequency in azoospermic with respect to normozoospermic group of patients with diversified ethnic backgrounds will be men in one study [106] while other studies revealed no dif- valuable to reinforce the role of USP26 gene in male ferences in the distribution of FSHR polymorphisms between infertility. normal and infertile men. Additionally, a FSHR SNP has been discovered that may affect the activity of the gene [21]. Balkan The TAF7L gene mutations et al., [105] investigated the genetic screening for SNPs at nucleotide position 29 in the core promoter region and codon TAF7L (transcription initiation factor TFIID subunit7-like) is 680inexon10oftheFSHR gene and the effect of the serum a member of TAFs that has an important role in transcription levels of FSH on male infertility in Southeast Turkey. They process. In humans, the TAF7L gene is an X-linked and single- suggested that the FSHR haplotype does not demonstrate any copy testis-specific gene that may be essential for mainte- association with different serum FSH levels. These different nance of spermatogenesis and a possible contributor to male results suggest that an in-depth analysis of role of LHR and infertility [112]. Recently, a number of publications have FSHR genes in male infertility is warranted. reported the importance of various TAFs genes such as TAF7L and TAF4b in mouse spermatogenesis. A study by Falender The USP26 gene mutations et al., [113]showedthatTAF4b (a gonad-specific subunit of TFIID) is required for the maintenance of spermatogenesis in The ubiquitin-specific protease-26 (USP26) gene, located on the mouse. In a knockout model study by Cheng et al., [112], human X chromosome (Xq26.2), is expressed throughout the they discovered that disruption of TAF7L gene in mice results 1124 J Assist Reprod Genet (2014) 31:1115–1137 in reduced sperm count and motility. They proposed that its organelles functional in oxygen consumption, unequivocally counterpart, the human TAF7L gene may also play a similarly demonstrating that motility depends on the mitochondrial important role in spermatogenesis. Because of the hemizy- activity. It has been recently demonstrated that in some gous state of the X chromosome in men, mutations in TAF7L asthenozoospermic patients the sperm midpiece was signifi- could potentially cause oligozoospermia in humans. On the cantly shorter than in normal subjects [126]. This finding other hand, Akinloye et al., [114], reported that TAF7L gene could be probably linked to defects in the energy producing mutations are rare and are not a common cause of spermato- machinery that is required to drive motility. Some studies have genic failure in normogonadotrophic nonobstructive azoo- revealed that spermatogenesis is associated with a drastic spermia patients. Although the TAF7L gene is highly poly- reduction in mtDNA content, to about a tenth of its initial morphic, most of them were not significantly associated with value, occurring mainly during spermiogenesis [127]. This gonadal dysfunction. In addition, they discovered that SNPs at result reinforces the idea that asthenozoospermia patients have exon 13 may be a risk factor for an infertile phenotype if defects in the sperm tail formation or energy production for combined with additional polymorphisms or mutations. These sperm motility. The change of sperm mtDNA copy number is findings warrant additional research to elucidate the role of a very important event throughout spermatogenesis, fertiliza- TAF7L gene as a molecular cause of male infertility. tion and embryogenesis. However, in one study by May- Panloup et al., [122], they reported that the mtDNA with deletions was significantly higher (up to 28 times) in sperm samples of poor quality than in normal sperm samples. The mitochondrial DNA (mtDNA) and male infertility Recent reports have shown that mtDNA point mutations or multiple deletions, mtDNA single nucleotide polymorphisms Significant strides in research have been made on the role of (SNPs) and mtDNA haplogroups can greatly influence sperm the mitochondria and its genome on male infertility [115]. quality and are associated with asthenozoospermia or Beside nuclear chromosome deficiencies that have profound oligoasthenozoospermia [128–138]. Among the mitochondri- effects on male fertility, there is increasing evidence that al deletions observed, the deletion of 4977 bp was the most mitochondrial DNA (mtDNA) anomalies in sperm may lead prevalent and abundant one. Kao et al., [132], for the first time to male infertility. Mitochondria play a critical role in energy reported that a 4977 bp mtDNA deletion is associated with metabolism by containing oxidative phosphorylation ele- diminished fertility and motility of human sperm and that the ments and enzymes (OXPHOS) [116]. There are 70–80 mito- highest frequency of occurrence of the deletion coincided with chondria in the midpiece of human mature spermatozoa that reduced sperm motility. Further studies have analyzed sperm serve as a large source of energy (ATP) for the flagellum to mtDNA deletions by long-range PCR techniques and found move around in the early stages of fertilization [117]. As the two other novel types of 7345 and 7599 bp large-scale dele- mtDNA genes encode several polypeptides particularly for the tions in the mtDNA of spermatozoa with poor motility [125]. OXPHOS, mtDNA damages cause deficiency in ATP produc- Although these three large-scale deletions were found to occur tion [118, 119]. The mitochondrial machinery plays a key role either alone or in different combinations, the 4977 bp deletion in the energy production and maintenance of spermatozoa is the most frequently (affecting 40 % of patients with mito- motility and is proposed to be one of the major determinants chondrial myopathy) occurring deletion in spermatozoa with of male fertility [120]. poor motility [125]. A higher percentage of multiple deletions, Substantial evidence has associated deficiencies of human 4977-bp, 7599-bp and 7491-bp of mtDNA in spermatozoa of sperm mitochondria with sperm dysfunction and poor sperm patients with poor sperm quality have also been recently quality [121, 122]. Asthenozoospermia or reported [136]. Other studies have confirmed the persistence oligoasthenozoospermia has been reported previously in pa- of multiple deletions in normozoospermic samples [139]. The tients suffering from typical mtDNA diseases [123, 124]. ATPase6, ATPase8 and COII genes deletions have been re- Other studies have shown a correlation between the quality ported previously in mtDNA of 49 asthenozoospermia pa- of the sperm and the functionality of the respiratory chain in tients [135]. However, there was a greater accumulation of sperm mitochondria [125]. A direct and positive correlation multiple deletions present when the sperm quality was poor, between sperm motility and mitochondrial enzymatic activi- with oligoasthenoteratozoospermic men harboring far more ties has been demonstrated suggesting that motility largely mtDNA deletions. dependsonenergyproductionbymitochondria[120]. In an Recent studies have shown that mutations in mitochondrial interesting study by Carra et al., [120], they investigated genes such as COX II, ND1, ATPase6and8disrupttheATP sperm mtDNA from idiopathic oligoasthenozoospermic pa- production and effect spermatogenesis and sperm motility. In tients, with different sperm motility and sperm concentrations, a study by Guney et al., [119], they investigated the relation- by testing motile and non-motile sperm of the same individ- ship between the mtDNA genes (ATPase6, Cytb and ND1) ual. Their results suggested that only motile sperm have and male infertility. They found 13 point mutations in the J Assist Reprod Genet (2014) 31:1115–1137 1125

ATPase6 gene, of which three were novel and 8 caused amino production of mitochondria ROS due to incomplete reduction acid changes. In addition, they examined for the first time the of oxygen. In addition to mitochondrial inner membrane association of Cytb gene mutations with infertility and detect- potential, ROS induces mtDNA strand breaks and large- ed 20 point mutations in the Cytb gene, all of which were scale deletions. The large-scale deletions result in complete statistically non-significant. About 8 mutations in ND1 gene removal or truncation of some structural and tRNA genes of were also observed in their study. Holyoake et al., [134] mtDNA and results in male infertility [147]. showed that point mutation in the ATPase6 gene at 9055 was commonly seen associated with poor sperm quality. This substitution involves a G to A transition within the ATPase6 Epigenetic modifications and male infertility gene, changing an alanine to threonine. In their study, about 10.7 % of men with poor semen quality were shown to have The recent advent of new technologies has spurred research the G9055A substitution, but only 1.3 % of men with normal on the role of epigenetics in spermatogenesis and male infer- fertility had the same substitution. Furthermore, a T8821C tility. Epigenetics refer to process of gene regulation devoid of point mutation has been found in the ATPase6 gene of severe- changes in DNA sequence and comprise of DNA methylation, ly oligospermic men with immature spermatids. In another posttranslational histone modifications or chromatin remodel- study, the T9098C transition was detected only in infertile ing [148]. Recently, hyper methylation of promoters of several cases, and the finding of this mutation was statistically signif- genes including MTHFR, PAX8, NTF3, SFN, HRAS, icant when compared to controls [138, 140]. Thangaraj et al., JHM2DA, IGF2, H19, RASGRF1, GTL2, PLAG1, D1RAS3, [141] also established that mitochondrial mutations in sperms MEST, KCNQ1, LIT1 and SNRPN have been reported to be can cause low sperm motility that is not related to infertility. associated with poor semen parameters or male infertility They observed 10 SNPs in the ATPase6 gene in the sperm [149]. DNA of an oligoasthenoteratozoospermic man. They found DNA methylation is one of the best-characterized epige- that these mutations were associated with low motility sperm netic processes and involves the addition of a methyl group to but otherwise did not impair fertility. In another study by the 5′ position of the cytosine nucleotide, typically occurring Spiropoulos et al., [142] they carried out semen analysis on in a CpG dinucleotide. This reaction is catalyzed by a group of a male harbouring the A3243G mtDNA mutation and showed termed DNA (cytosine-5) methyltransferases that high levels of mutant mtDNA strongly correlated with (DNMTs) [150]. CpG islands have been found near promoters low sperm motility. Folgero et al., [131] also previously noted andhavebeendocumentedtoplayanimportantrolein reduced sperm motility in a patient with mtDNA disease due regulating gene expression [149]. It has been reported that to the A3243G mtDNA mutation. Two specific point muta- hyper methylation of DNA in CpG islands is associated with tions in the region of mtDNA genome from COX I (complex gene inactivation, while hypo methylation being associated IV) and ND 5 (complex I), at nt 9055 and nt 11719 are with gene activation [151]. Improper DNA methylation of associated with poor semen quality parameters in 11 and various genes has been implicated in abnormal semen param- 12 % of cases respectively. T26248G-transversion mutation eters, as well as several instances of male infertility. in exon7 of the putative methyltransferase Nsun7 gene has Houshdaran et al., [152] demonstrated for the first time, that been recently reported to lead to low sperm motility [143]. poor sperm concentration, motility and morphology were Nuclear gene defects may also result in mitochondrial associated with broad DNA hyper methylation across a num- disorders by predisposing the cell to multiple mtDNA dele- ber of loci including PLAG1, DIRAS3, MEST, PA X8, NTF3, tions. Recently, the nuclear encoded proteins including SFN and HRAS. They suggested that the underlying mecha- mitochondrial-specific DNA polymerase gamma (POLG) nism for these epigenetic changes may be improper erasure of [144] and glutathione S-transferase M1 have been reported DNA methylation during epigenetic reprogramming of the to be involved in male infertility [145]. POLG is responsible male germ line. In another study by Khazamipour et al., for replication and repair of the mtDNA. Mutations in the gene [153], 53 % of men with nonobstructive azoospermia revealed for the catalytic subunit of POLG have been shown to be a hypermethylation of the MTHFR promoter, while none of the frequent cause of mitochondrial disorders and sperm dysfunc- men with obstructive azoospermia exhibited hypermethyla- tion [146]. Hence, mitochondrial respiratory chain function tion of this gene promoter, suggesting that MTHFR hyper- depends on the coordinated gene expression of both the mi- methylation is a specific epigenetic aberration that may spe- tochondrial and nuclear genomes and a mutation in either cifically contribute to certain types of male infertility. In genome may lead to defective respiratory function of addition, Wu et al., [154] in a study reported that hyper- mitochondrion. methylation of MTHFR gene promoter in sperm was associ- Mitochondria is a major intracellular source of reactive ated with idiopathic male infertility. Abnormal DNA methy- oxygen species (ROS). It is believed that mtDNA mutation lation of H19 and MEST imprinted genes has also been shown may impair electron transport chain resulting in enhanced to be associated with oligozoospermia [140], suggesting that 1126 J Assist Reprod Genet (2014) 31:1115–1137 spermatogenesis may be particularly vulnerable to changes in information to the embryo to guide it through development. the methyl pool brought about by deficiency in MTHFR The lack of proper DNA packaging in sperm has been enzyme. Hammoud et al., [155] examined CpG methylation associated with infertility in mice [160]. During the chroma- patterns in infertile (oligozoospermic and abnormal prot- tin re-packaging in spermatogenesis process, about 85 % of amine) and fertile donors at seven imprinted loci including the histones are replaced by protamines [161]. In the initial LIT1, MEST, SNRPN, PLAGL1, PEG3, H19,andIGF2.Atsix stages of spermiogenesis, histones are hyper acetylated and of the seven imprinted genes, the overall DNA methylation undergo other modifications [162]. At the final stage of patterns were significantly altered in both infertile patient spermatogenesis, the nucleosomal structure is progressively populations. They demonstrated a link between abnormal disassembled, then replaced by transition proteins (TNPs) spermatogenesis and abnormal methylation of genes. Contra- and finally by protamines [163]. The incorporation of prot- dictory reports demonstrated high methylation at the H19/ amines into sperm chromatin induces DNA compaction that IGF2 ICR1 locus and low methylation at the MEST locus in is important for the formation of spermatozoa. However, it is normozoospermia in one study, while another study revealed known that protamines are phosphorylated before binding to low methylation of the H19/IGF2 ICR1 locus and high meth- DNA and that substantial dephosphorylation takes place ylation of the MEST locus in association with low sperm concomitant with nucleoprotamine maturation. Indeed, mu- concentrations [156]. Similarly, Boissonnas et al., [157]found tation of the calmodulin-dependent protein kinase Camk4 that many patients with teratozoospermia and that phosphorylates protamine 2, results in defective sper- oligoasthenoteratozoospermia exhibited hypo methylation at miogenesis and male infertility [164]. At fertilization, the variable CpG islands at the H19 locus. However, recent stud- mature sperm cells are packaged densely with protamines, ies have shown that hypermethylation at MEST was more whereas maternal genome is packaged with histones. Sub- strongly linked with poor sperm quality than hypo methyla- sequently, upon fertilization, the highly compact tion at H19/IGF2 ICR1 [148]. They suggested that sperm nucleoprotamine structure must be unpacked and from infertile patients, especially those with oligospermia, reorganized into a nucleosomal structure [165]. Recent stud- may carry a higher risk of transmitting incorrect primary ies have shown that some epigenetic errors in these process- imprints to their offspring, highlighting the need for more es are a possible cause of male infertility. Both protamines 1 research into epigenetic changes, when considering ART. and 2 are essential for sperm function, and the Post-translational histone modifications, another compo- haploinsufficiency of either P1 or P2 results in a reduced nent of epigenetic process is essential during many cellular amount of the respective protein, abnormalities in the struc- processes including mitosis and spermatogenesis. The com- ture of the chromatin, DNA damage and infertility [148]. pactness of DNA is determined by histone proteins that are Furthermore, it is known that the optimal protamine-1 to bound to DNA [148]. Histone methylation, one of the most protamine-2 ratio (P1/P2 ratio) is critical and well regulated prevalent modifications, is catalyzed by histone methyltrans- [166]. Indeed, the P1/P2 ratio in fertile men ranges from 0.8 ferase enzyme (HMTases) and is generally associated with to 1.2 and deviation from this ratio has been shown to lead gene silencing. Histone acetylation, modulated by both his- to infertility [167]. A change in either direction of this ratio tone acetyl transferases (HATs) and histone deacetylases adversely affects semen quality, DNA integrity and fertility (HDACs), is associated with increased levels of transcription. in men. Patients with abnormally depressed or elevated P1/ HATs activate gene expression, while HDACs inhibit gene P2 ratios are characterized by poor sperm concentration, expression. Recent research has identified a critical role for the motility and morphology as well as decreased fertilization JHMD2A (Jumonji C-terminal containing histone capabilities. Impaired spermatogenesis has been reported to demethylase 2A) histone demethylase in male infertility and be associated with aberrant H4 acetylation [168]. A study by spermatogenesis [158, 159]. Using knockout mice as models, Sonnack et al., [169] showed that men exhibiting qualitative this study identified a critical role for JHMD2A in the regula- or quantitative infertility have significantly decreased levels tion and expression of two genes, protamine 1 (Prm1)and of histone H4 acetylation associated with impaired sper- transition nuclear protein 1 (Tnp1) involved in the condensa- matogenesis. Hyperacetylation of histone H4 is required in tion and proper packaging of chromatin in the male sperm. the transition from histones to protamines. This step de- Chromatin remodeling is a process in which ATP- creases the affinity of the interaction between the sperm dependent chromatin remodeling complexes (such as SWI/ histones and DNA to allow the exchange for transition SNF, ISW1 and MI-2) use energy to alter the location and proteins to occur [149]. H4 hyperacetylation was also ob- structure of nucleosomes [106]. The remodeling process served in infertile men exhibiting SCOS [150]. A significant activates or represses gene expression as required for proper difference in transcript expression of chromatin remodeling development and maturation of gametes and proper meiosis factors between normal spermatogenesis and round sperma- [149]. It is a critical step in gamete development, and the tid maturation arrest has been found in a previous study, compacted structure of the chromatin transmits vital suggesting impaired epigenetic information and aberrant J Assist Reprod Genet (2014) 31:1115–1137 1127 transcription during sperm development. This could repre- analysis. An in-depth spatial and temporal analysis of gene sent one possible reason for the developmental arrest of expression could be useful to determine the genes that are round spermatids [170]. involved in each stage of the process [17]. Substantial evi- Genetic imprinting, namely the expression of alleles in a dence points to the presence of remarkably different RNA sex-specific manner, is most often due to the differential populations in mature sperm in fertile versus infertile men methylation in the CpG islands in one of the alleles [148]. It [139]. HSPA2 is correlated with sperm maturity, function and determines which genes from the paternal and maternal ge- fertility, and its dysfunctional expression results in abnormal nomes are expressed in the embryo and is critical for normal spermatogenesis. A study by Li et al., [174]demonstrated development [171]. Several loci are known to be imprinted. lower levels of HSPA2 expressed in sperm in oligoteratozoos- For instance, in humans, fetal spermatogonia seem to be permic men compared to normozoospermic controls. Yet an- mostly unmethylated at H19 differentially methylated regions, other preliminary study [174] revealed significantly lower although spermatogonia in adult testis demonstrate significant levels of BDNF and NGF receptor TrkA mRNAs in human methylation in this region [148]. Imprinted genes, including sperm from oligoasthenozoospermic men compared to normal the paternally imprinted GTL2 and H19 loci, have been pre- controls. Glycoprotein subunit 130 (GP130), upon binding to viously examined by Kobayashi et al., [171]in97infertile IL-6, is a known regulator of sperm motility. Low levels of men. The importance of during spermato- GP130 mRNA and protein have been reported in genesis has been reinforced by the association of decreased asthenozoospermic men when compared to normozoospermic methylation of the paternal IGF2⁄H19 imprinting control re- men [175]. Guo et al., [176] compared the levels of gion 1 (ICR1) and GTL2 imprints in spermatozoa of men with DDX4/VASA transcript, an RNA binding protein/helicase disturbed spermatogenesis. In another study by Poplinski that is essential for germ cell development, between normo- et al., [156], fertile men had a high degree of IGF2⁄H19 zoospermic men and patients with oligozoospermia. VASA ICR1 and a low degree of MEST methylation. Low sperm transcript and protein levels were significantly decreased in counts were clearly associated with IGF2⁄H19 ICR1 hypo the sperm of oligozoospermic men, suggestive of the fact that methylation and, even stronger, with MEST hyper methyla- VASA is potentially associated with pathogenesis in some tion. These results suggest that idiopathic male infertility is selective subtypes of male infertility. strongly associated with imprinting defects at IGF2⁄H19 ICR1 and MEST, with aberrant MEST methylation being a strong miRNAs in sperm indicator for sperm quality. ART techniques such as ICSI and round spermatid injection (ROSI) may increase the incidence Awareness of the presence of myriad groups of RNAs and of imprinting disorders and adversely affect embryonic devel- their elusive functions in spermatogenesis, fertility and early opment by using immature spermatozoa that may not have embryo development, have led to increased focus on small established proper imprints or global methylation yet. regulatory RNAs and their function in these processes. Small Andrologists have voiced concern about concealing reproduc- regulatory RNAs are non-coding RNAs that regulate gene tive defects through ART that might have negative conse- expression at the transcriptional, post-transcriptional or quences at the epigenetic level. through chromatin remodeling [177]. It has been established that human spermatozoa comprises of miRNAs (7 %) piRNAs (17 %) and repeat-associated small RNAs (65 %) Diversity of RNAs in sperm [178]. miRNAs are single-stranded noncoding RNAs (20–25 nu- The diverse RNA populations present in the spermatids are cleotides) that are mostly expressed in a tissue-specific fash- produced either throughout early spermatogenesis or during ion, and down regulate gene expression through their interac- spermiogenesis and serve as a historical record of spermato- tion with the 3′ untranslated region (UTR). Approximately genesis. The highly condensed sperm nucleus is transcrip- one-third of human genes are regulated by miRNAs [179]. tionally inert and contains mRNA, antisense RNAs, and They are implicated in the regulation of many important miRNAs that have been transcribed prior to inactivation. biological pathways such as proliferation, apoptosis, and dif- Despite the inert status of the sperm RNAs, the delivery of ferentiation in various species, but a large number of them sperm mRNAs to the oocyte at fertilization is speculated to (about 68 %) show a tissue-specific expression [180]. They be important in the early development of the zygote and are expressed in various testicular cell populations as well as embryo [172, 173]. epididymis, indicating a critical role in the various steps of the Recent advances in technology have been instrumental in highly organized process of spermatogenesis, sperm matura- deciphering the role of sperm RNAs in development and tion and maintenance of male fertility. More than 200 disease. Transcriptomics involves profiling of gene expression miRNAs have also been identified in the human epididymis using microarrays, RT-PCR and in situ hybridization (ISH) and this could suggest their potential involvement in 1128 J Assist Reprod Genet (2014) 31:1115–1137 regulating the physiological functions of sperm maturation unnecessary for spermatogonial stem cell renewal and mitotic and morphogenesis [181]. Members of the miR-888 cluster proliferation, but is required for germ cell differentiation (miR-890, miR-891a, miR-891b, miR-892a and miR-892b) through the meiotic and haploid phases of spermatogenesis. are significantly more abundant in the corpus/cauda regions of Of relevance to the function of DICER1and DROSHA in the epididymis, indicative of a prominent role of the miR-888 miRNAs biogenesis and spermatogenesis, it has also been cluster in regulating physiological functions of the human demonstrated that mutations or single nucleotide polymor- epididymis. In another instance, expression of spag8 (sperm phisms (SNP) in Dicer1 and Drosha is associated with male associated antigen 8) mRNA transcript encoding for a human infertility. A mutation in Dicer1 or Drosha may cause global sperm membrane protein (hSMP-1) negatively correlates with alterations of miRNA expression and result in the up- the expression of miR-892a in the epididymis. regulation of their target genes, which in turn could affect Other recent reports have also highlighted the importance other interacting factors such as the miRNA 17–92 cluster, of miR-18 in the control of heat shock factor 2, a transcription leading to apoptosis of spermatozoa and abnormal semen factor that is required for normal spermatogenesis in mouse quality [195]. In a study by Qin et al., [196], they reported a [182]. miR-34c and miRNA34b are important players in the significant association between Dicer1 and Drosha SNP with later steps of spermatogenesis in germ cells [233] and differ- abnormal semen parameters and idiopathic male infertility. entiation of male germ cells [183]. The role of miR-122a that The genetic variants of rs12323635 (Dicer1), observed in regulates Tnp2, a testis-specific gene involved in chromatin Han-Chinese population were also associated with idiopathic remodeling during spermatogenesis cannot be underscored male infertility. Additionally, men with the rs642321TT ge- [184, 185]. Additionally, miR-17–92 cluster functions in the notype (Drosha) may have a higher risk of oligozoospermia. regulation of apoptosis and -1 transcription factor during The results showed that variants of Dicer1 and Drosha may normal spermatogenesis [186]. In a recent study, high levels of modify the risk of abnormal semen parameters, and could expression of specific miRNAs was observed in low fertility result in male infertility. Due to their functional diversity and bulls, which could suggest that miRNAs might be responsible the increasing complexity of miRNAs, it is clear that we have for down regulating gene expression and thereby, regulate just touched the tip of the iceberg and a lot more needs to be proteins that have important roles in fertilization and early done to understand the diverse and crucial roles of miRNAs embryonic development [187]. Other studies revealed signif- and other DICER-dependent RNA pathways in male germ icant differences in the expression of miRNAs between im- cell biology. mature and mature individuals, suggesting miRNAs have a Recent studies have also revealed the presence of miRNAs role in regulating spermatogenesis [188, 189]. Special men- in serum, plasma, semen and other body fluids. The produc- tion needs to be made of miR-322, miR-323, miR-372 and tion of miRNAs in body fluids and their biogenesis patterns miR-373 that are presumed to be novel oncogenes that partic- are closely correlated with various diseases [197, 198]. Hu- ipate in the development of human testicular germ cell tumors man semen contains several species of miRNAs that can be [190]. readily detected by RT-qPCR and used as a diagnostic tool for Even deletions or mutations in miRNA processing machin- semen stain identification. It is assumed that miRNAs may ery of Dicer1 and Drosha can cause impaired spermatogene- leak either passively or actively from apoptotic or broken sis and male infertility. Primary miRNAs molecules exist in sperm cells, germ cells or from cells of accessory glands into intronic regions and are produced by RNA Pol II, then further seminal plasma [198]. The binding of miRNAs with complex processed through a complex process in nucleus (by the organic molecules and the RNA-macromolecular complex microprocessor machinery known as DROSHA) and subse- render them more or less resistant to RNase degradation and quently transported to cytoplasm (by an endonuclease DIC- they are stable in seminal plasma. Additionally, fertile and ER1) [191, 192], leading to mature miRNAs production. infertile patients have some specific miRNAs in their semen DICER1and DROSHA are also reported to play crucial roles that can be used as potential biomarkers to distinguish be- in the normal function of somatic nursing cells of the semi- tween them. Since reproductive disorders-associated miRNAs niferous epithelium and the process of normal spermatogene- can be identified in seminal plasma, a non-invasive semen test sis. Many studies have highlighted the critical roles of DIC- could be developed to diagnose male infertility. Thus, it is ER1 and DROSHA in male infertility. For instance, knockout reasonable to speculate that the concentrations of miRNAs in of Dicer1 in mice Sertoli cells or male germ cells leads to seminal plasma may provide some useful information about infertility due to impaired spermatogenesis, progressive tes- gene expression in the male reproductive system and may ticular degeneration and both meiotic and spermiogenic de- represent a new source of novel, minimally invasive bio- fects [193]. Papaioannou et al., [194] also reported that abla- markers of male infertility. tion of Dicer1 in Sertoli cells leads to infertility due to the Recent studies also reported a significant difference in the complete absence of functional spermatozoa and progressive concentration of some specific miRNAs in seminal plasma of testicular degeneration. Another study indicated that Dicer1 is infertile men [198, 199]. For instance, Wang et al., [199], J Assist Reprod Genet (2014) 31:1115–1137 1129 observed that 7 special miRNAs (miR-34c-5p, miR-122, miR- Power of proteomics in male infertility 146b-5p, miR-181a, miR-374b, miR-509 –5p and miR-513a- 5p) are markedly decreased in semen of azoospermia but The proteomics techniques allows for the measurement of increased in asthenozoospermia. They revealed a strong rela- protein levels and accurately determines the changes in all tionship between the seminal plasma miRNAs and male in- proteins expressed and translated from a single genome in a fertility, suggesting that the seminal plasma concentrations of tissue or cell. Proteins are identified with two-dimensional these miRNAs can accurately distinguish infertile patients electrophoresis (2-DE) and mass spectrometry (MS) tech- from fertile controls. These recent discoveries of differential niques, and the data are used to create the profiles of proteins expression of miRNAs in testis have generated a new ap- in the given sample [260]. Proteomic technology serves as a proach, miRNAs profiling, to reveal functional information diagnostic tool to probe the pathology and idiopathic causes of of miRNAs relevant to this disorder. Needless to say, miRNA male infertility, and the seminal plasma and sperm proteins research is an emerging and exciting field that holds promise identified can also be validated by Western blot [207]. In for improvements in diagnosis and treatment of male addition, comprehensive studies of the sperm or seminal pro- infertility. teome can be instrumental in the identification of differences in protein expression between normal and abnormal sperm piRNAs and male infertility populations, enabling the identification of potential novel biomarkers for diagnosis, prognosis and development of ther- Another newly identified class of small RNAs deemed to apeutic options to infertile men. regulate spermatogenesis and male infertility, is that of During the spermatogenesis process and sperm maturation piRNAs. They are the largest and most complex class of in the epididymis and post-ejaculatory capacitation in the small non-coding RNAs that interact with piwi-family pro- female reproductive tract, post-translational modifications teins and include MIWI, MIWI2, HIWI, PRG-1, PRG-2 and (PTM) are critical for the functionality of spermatozoa MILI [200]. Not only are they DICER-independent, they are [208–210]. PTMs that are formed by glycosylation, methyla- also distinct from miRNAs in their length (24–30 nucleo- tion, or phosphorylation are able to change the functional tides) and expression patterns. They are expressed particu- properties of spermatozoa and seminal plasma proteins larly in pachytene spermatocytes and round spermatids dur- [211]. Thus, identification of differentially expressed proteins ing spermatogenesis, indicative of a role of these small and their modification patterns between fertile and infertile RNAs in development and protection of male germ cells patients could provide an opportunity to study candidate genes from invasive transposable elements [201]. The piRNAs and other abnormalities in search for mutations. repress retrotransposons and regulate at the post- Proteomic profiling studies revealed that human sperm and transcriptional level. The roles of piRNAs in spermatogene- seminal plasma contain a large number of proteins such as sis are supported by the well-established functions of their heat shock protein (HSP), fibronectin, semenogelins, and partner, the Piwi proteins. Recent evidence indicates that the laminin, as well as enzymes such as serine protease, piwi subfamily proteins are essential for stem cell self- prostate-specific antigen (PSA), prostatic specific acid phos- renewal, the development of male germ cells and spermato- phatase (PSAP), and creatine kinase [212]. Additionally, an- genesis [202]. For instance, spermatogenesis is arrested at timicrobial proteins including lactoferrin, protease inhibitors the pachytene spermatocyte stage in Mili-knockout mice such as R-1-antitrypsin, and transport proteins including albu- [203]. In Miwi2-knockout mice, spermatogenesis stops with min are also abundant in the semen [213]. The large number of meiotic arrest at the leptotene stage [204]. Germ cells in identified proteins indicates the complex composition and Miwi-deficient mice testis undergo meiosis without elongat- function of human sperm. Wang et al., [199]identified4675 ed spermatids or mature spermatozoa production [205]. Fur- human sperm proteins, of which 227 were testis-specific thermore, the small non-coding RNAs Nct1 and Nct2 have proteins. Approximately 403 different proteins have also been recently been suggested to be piRNA precursors and are identified from the isolated sperm nuclei, the most prominent expressed particularly in pachytene spermatocytes [206], among them being the histones. Majority of sperm proteins which also points to a role of piRNAs in the regulation of have been functionally categorized as those involved in sperm the meiotic stage in spermatogenesis. Although, Nct1 and movement and structural organization (34 %), energy and Nct2-deficient mice display a decrease in a small cluster of metabolism (27 %), stress response, protein folding and pro- piRNAs located on chromosome 2, it does not affect mouse tein turnover (22 %), signaling and transport (8 %) and anti- spermatogenesis or fertility, suggesting that those piRNAs oxidantactivity(6%)[214]. The others are essential in the located on chromosome 2 are necessary to maintain trans- capacitation of the spermatozoa, modulation of the immune poson silencing [202]. Thus, it is likely that piRNAs are responses in the uterus, formation of the tubal sperm reservoir potentially involved in regulating the processes of meiosis and finally in both the sperm-zona pellucida (ZP) interaction and post-meiosis of male germ cell development. and sperm and egg fusion. 1130 J Assist Reprod Genet (2014) 31:1115–1137

Differential profiling studies between fertile and infertile cells of idiopathic infertile patients. Therefore, proteomics patients have demonstrated that the identification of differen- studies will allow for a better understanding of PTMs and tially expressed proteins could help elucidate mutations in offer the opportunity to identify proteins that are differentially candidate genes as well as identify biomarkers that can, in expressed in abnormal semen samples and potentially in- turn, help clinicians determine certain peptides or metabolites volved in idiopathic male infertility. that may be linked to male infertility [215]. For instance, Sharma et al., [212] have reported that there is significant difference in protein expression between men with normal and Diagnosis of male infertility abnormal sperm. In a study detailing oxidative stress, Sharma et al., also demonstrated that the increased expression of To date, the diagnosis of male infertility has been primarily several proteins especially histone cluster 1H2ba based on physical exams, post-ejaculatory urine sample, (HIST1H2BA), mitochondrial malate dehydrogenase precur- blood tests and traditional semen analysis, with a strong sor (MDH2), transglutaminase-4 (TGM4), glutathione emphasis on the assessment of semen volume and sperm peroxidase-4 isoform A precursor (GPX4), glutamine synthe- concentration, motility and morphology. But mounting ev- tase (GLUL), heat shock proteins (HSP9 0B1 and HSPA5) in idence suggests that routine evaluation of semen quality is the ROS+ patients, suggests that these proteins can serve as insufficient to reflect the overall quality of sperm [213]. potential biomarkers of oxidative stress [216]. Thacker et al., Large-scale molecular genetics analyses have indicated that [217], found that four unique proteins including semenogelin ~3000 genes are involved in male reproduction [173]. II precursor, prolactin-induced protein, clusterin isoform 1, Detailed mechanistic studies of these genes have aided in and PSA isoform 1 preproprotein were predominantly present the development of diagnostic tests to test male infertility in the semen of healthy men. In another study based on 2D and therapeutic strategies to overcome infertility, particular- SDS–PAGE followed by MALDI MS/MS analysis, Siva et al., ly in men undergoing the ICSI procedure. To name a few, [214] confirmed significant changes in intensity of protea- testicular mapping, genetic analysis at the chromosomal some alpha-3 subunit in asthenozoospermic samples when level (karyotyping), androgen receptor gene mutations, CF compared with normozoospermic controls. Significant posi- test and Y chromosome micro deletion analysis are being tive correlation was found between proteasome alpha-3 sub- used routinely in diagnosis of male factor infertility [15]. In unit levels and rapid, linear progressive motility of the sper- addition, some molecular cytogenetic methods such as matozoa. In a preliminary study, Martinez-Heredia et al., comet assay, sperm-chromatin-dispersion assay (SCD), [218] identified 17 proteins, particularly cytoskeletal actin-B, transferase-mediated dUTP nicked labeling (TUNEL), or annexin-A5, cytochrome C oxidase-6B, histone H2A, sperm chromatin structure assay (SCSA) have been devel- prolactin-inducible protein and precursor, calcium binding oped for the detection of DNA damage [4]. Although, the protein-S100A9, clusterin precursor, dihydrolipoamide dehy- pre-implantation genetic diagnosis (PGD) technology is not drogenase precursor, fumarate hydratase precursor, heat shock a routine method in the evaluation of male infertility, it protein-HSPA2, inositol-1 mono phosphatase, 3-mercapto- could potentially prevent the transmission of genetic anom- pyruvate sulfur transferase, dienoyl-CoA isomerase precursor alies to the offspring [4]. These tests can help identify and proteasome subunit-PSMB3, in asthenozoospermic DNA fragmentation, chromosomal defects, or the possibil- sperm samples when compared with the normozoospermic ity of genetic diseases that can be passed on to children. individuals. Similarly, Zhao et al., [219], found 10 proteins to Currently, Y chromosome micro deletions are analyzed by be differentially expressed in these samples when compared PCR-screening of STSs from different regions of the AZF with the normozoospermic individuals. Li and Zhang [211] on the long arm of the Y, as well as by using DNA probes reported that HSP70-2, PLC, GPX4, β-tubulin and GAPDHS of the genes (Fig. 1). In the recent years, it has become are associated with sperm motility, and thereby male fertility. possible to detect the incidence of chromosomal abnormal- They showed these proteins are potential markers for the ities using a variety of high-powered polymerase chain mechanisms of infertility or radiotherapy, because their ex- reaction (PCR) techniques and multi-colour fluorescent in pression pattern is changed in these cases. β-tubulin is essen- situ hybridization (FISH) analysis [4]. Before patients can tial for sperm axoneme migration, flagellar movement and be subjected to ICSI, FISH-sperm studies are necessary to regulation. GAPDHS is also related to sperm energy supply. understand whether there is a genetic cause for male infer- Sperm proteome studies on asthenozoospermia samples have tility. If genetic abnormalities are suspected in either part- also revealed that some component of the proteasome com- ner, counseling is recommended. Recently, further tests, plex is differentially expressed, suggesting an importance of such as ROS determination and chromatin fragmentation, the proteasome complex in sperm motility [220]. These results have also been proposed for research purposes [115]. In suggest the validity of the proteomic approach to identify light of recent proteome and transcriptome profiling analy- proteins with altered amounts in the human semen and sperm ses, researchers strongly advocate for additional new J Assist Reprod Genet (2014) 31:1115–1137 1131 markers of fertility or infertility that could include sperm References and seminal plasma proteins, complex population of RNAs including miRNAs and other small noncoding RNAs that 1. Vogt PH. Molecular genetic of human male infertility: from genes to are retained in mature sperm. new therapeutic perspectives. 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